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Abstract: One of the most essential systems applied to the eradication of bovine tuberculosis by Mycobacterium bovis is the epidemiologic surveillance of animals slaughtered in abattoir by means of inspection and sample taking of lesions compatible with tuberculosis, confirming the existence of the disease through culture and molecular detection, which takes weeks before a result can be obtained. An interesting alternative is to develop high-throughput molecular systems for the direct detection of M. bovis on biological samples. In this sense, our research has developed a molecular detection system by means of a real-time based PCR process which is applied directly to bovine biological samples and it allows to differentiate between Mycobacterium tuberculosis complex, Mycobacterium avium complex and other atypical mycobacteria that are interesting from the veterinary point of view. The sensitivity was analyzed by applying a conventional extraction system based on guanidine thiocyanate and a robotized system based on the selective magnetic capture of mycobacterial DNA. The molecular detection system showed a high specificity and a detection threshold of only two to three genomes. The sensitivity depended on the DNA extraction system being used and on the kind of lesions on which it was used; the sensitivity ranged from 61.11% for samples with non-visible lesions to 80.64% for chronic lesions, with an average sensitivity of 73.87% when using the manual extraction system and between 27.77 and 74.19% (average sensitivity 47.74%) when using the automated robotic system. In conclusion, our multiplex real-time PCR assay represents a fully controlled, high-throughput diagnostic tool for the rapid detection of Myobacterium presence directly in animal clinical specimens, which could be a practical tool in the context of bovine tuberculosis abattoir surveillance programs and granuloma submission programs. [Copyright &y& Elsevier]

Abstract: This study was carried out to evaluate the role of wild artiodactyls as reservoirs of Escherichia coli O157:H7 for livestock and humans. Retroanal mucosal swabs samples from 206 red deer (Cervus elaphus), 20 roe deer (Capreolus capreolus), 6 fallow deer (Dama dama) and 11 mouflon (Ovis musimon), collected during the hunting season (autumn–winter) in South-western Spain, were screened. Samples were pre-enriched in modified buffered peptone water, concentrated by an immunomagnetic separation technique and cultured onto selective cefixime tellurite sorbitol MacConkey agar. Polymerase chain reaction (PCR) was used to detect the presence of genes coding O157 and H7 antigens and the virulence factors verocytotoxin, intimin and enterohaemolysin. Three E. coli O157:H7 isolates were obtained from red deer (1.5%). Two of them showed inability to ferment sorbitol and lack of β-d-glucuronidase (GUD) activity, however, the other strain investigated was an atypical sorbitol-fermenting E. coli O157:H7 with GUD+ activity. This is the first report pointing to red deer as a reservoir of E. coli O157:H7 in Spain. [Copyright &y& Elsevier]

Recreational hunting of indigenous wild artiodactyls has been one of the most lucrative and rapidly growing industries in Western Spain over the last five years. In the absence of careful ecological management, one consequence of the commercial exploitation of this natural resource has been the appearance of outbreaks of infectious disease; most notably bovine tuberculosis. From the outset of the study in 1997, we have observed a steady increase in prevalence of Mycobacterium bovis (M. boris) in both species reaching 1.74 (±0.17) in deer in 2002 and 2.32 (±0.24) in wild boar. The latter species seems to be most severely affected with pulmonary lesions appearing more chronic than those observed in deer. In this study, we describe the epidemiology of M. bovis in European wild boar (Sus scrofa) and Iberian red deer (Cerrus elaphus hispanicus) in Extremadura (W. Spain); a region where there are large areas of natural habitat for these species. [ABSTRACT FROM AUTHOR]

A molecular epidemiological approach was applied to establishing a possible role for the wild boar as a natural reservoir of Mycobacterium bovis in Sierra de Villuercas, Western Spain; an area free of farmed cattle and wild deer populations. Spoligo and VNTR typing were used over a three year period to study the epidemiological relationship between the occurrence of bovine tuberculosis (TB) in extensively bred Iberian pigs and indigenous wild boar. The 37 sampled wild boar showed different degree of calcified granulomatous lesions in retropharyngeal, mediastinal and pulmonary lymph nodes. The 25 sampled Iberian pigs showed calcified lesions, mainly in the respiratory tract. Lesions located in the mesenteric lymph nodes appeared secondarily. M. bovis was isolated from all affected animals. Twenty-five and 37 isolates of M. bovis were obtained from domestic pigs and wild boar, respectively. Our findings provide evidence that supports the possibility of cross infection between wild boar and domestic pig populations. This is contrary to the generally held belief that swine represent an epidemiological dead end host and play no role in the epidemiology of M. bovis. [Copyright &y& Elsevier]

Abstract: Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with cattle recognized as their main natural reservoir. The aim of this work was to determine the prevalence and the pheno-genotypic characteristics of STEC O157:H7 in a herd with 268 cattle of the fighting bulls breed (De Lidia breed) managed under extensive conditions in the South-West of Spain. Rectal–anal swabs of all animals were collected and examined for STEC O157:H7 by performing an immunomagnetic concentration and separation procedure combined with PCR, and the resulting isolates were characterized by both phenotypic and genotypic methods. Overall, STEC O157:H7 was isolated from seven animals (2.6%) in the herd. The PCR procedure indicated that all seven isolates displayed stx 2, eae-γ1, ehxA, O157 rfbE, and fliCh7 genes. They belonged to phage types 4 (one isolate) and 42 (two isolates), and four isolates reacted with typing phages but did not conform to a recognized pattern. Among the seven isolates there were five indistinguishable PFGE patterns and other two which differed only in⩽2 restriction fragments, supporting the existence of horizontal transmission among animals in the herd. The present study demonstrates that cattle managed under extensive conditions in Spain can excrete STEC O157:H7 with their faeces. To our knowledge this is the first isolation of this pathogen from De Lidia cattle. [Copyright &y& Elsevier]